Fucosylated oligosaccharides and process for their preparation

ABSTRACT

The present invention relates to a process for the preparation of oligosaccharides or oligosaccharide containing compounds, especially N-acetyl-chitooligosaccharides having a fucosylated monosaccharide. The invention also relates to novel oligosaccharides or oligosaccharide containing compounds, especially N-acetyl-chitooligosaccharides, which are fucosylated and optionally covalently bound to a carrier molecule.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/FI00/00803 which has an Internationalfiling date of Sep. 21, 2000, which designated the United States ofAmerica.

FIELD OF INVENTION

The present invention relates to novel fucosylated oligosaccharides oroligosaccharide containing compounds which are analogues to naturaloligosaccharides and which contain at least one fucosylatedmonosaccharide unit. The invention also relates to a process for thepreparation of such oligosaccharides or oligosaccharide containingcompounds.

BACKGROUND OF THE INVENTION

Fucosylated mammalian glycans have functions in fertilization (1), earlydifferentiation of embryo (2), brain development (3,4), and leukocyteextravasation (5,6). the α1-3/4fucosylated oligo- and polysaccharidesbeing conjugated to lipids and proteins or as free oligosaccharides suchas the oligosaccharides of human milk. The fucosylatedN-acetyllactosamines (Lewis x, Galβ1-4(Fucα1-3)GlcNAc and Lewis a,Galβ1-3(Fucα1-4)GlcNAc) and fucosylated N-acetyllactosdiamine (LexNAc,GalNAcβ1-4(Fucα1-3)GlcNAc) occur often as terminal sequences such asGalβ1-4(Fucα1-3)GlcNAcβ1-2Manα-, Galβ1-3)(Fucα1-4)GlcNAcβ1-2Manα-,GalNAcβ1-4(Fucα1-3(GlcNAcβ1-2Manα-,Galβ1-4(Fucα1-3)GlcNAcβ1-3(Galβ-/GalNAcα-,Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ- , Galβ1-4(Fucα1-3) GlcNAcβ1-4Manα-,Galβ1-3(Fucα1-4(GlcNAcβ1-4Manα-, andGalβ1-4(Fucα1-3)GlcNAcβ1-6Galβ-/GalNAcα-/Manα-. In the middle oflactosamine-type chains the Lewis x-sequences,-GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Gal are common, but also Lewisa-structures, Galβ1-3(Fucα1-4)GlcNAc, are present with a possibility forrepeating the sequence chain. In the free oligosaccharides found mainlyin human milk, the α1-3fucosylated epitope at the reducing end of thesaccharide is commonly lactose (Galβ1-4Glc) or its elongated/substitutedform such as Galβ1-4(Fucα1-3)Glc, Fucα1-2Galβ1-4(Fucα1-3)Glc,Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3(Glc, andGalβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc. Analogs of these could beuseful for studies of specificities of biological activities of thenatural mammalian α1-3/4fucosylated sequences.

The fucosylation step in the biosynthesis of these glycans isaccomplished by the family of α1-3/4fucosyltransferases (Fuc-Ts). Inman, at least Fuc-Ts III-VII and IX are expressed (7-9), andenzymatically active homologs are known in other animals and bacteria.

α1-3Fucosyltransferases transfer fucose to position 3 of GlcNAc or Glcresidues in Galβ1-4GlcNAc (LacNAc), GalNAcβ1-4GlcNAc (LacdiNAc) andGalβ1-4Glc (lactose) to synthesize the bioactive epitopesGalβ1-4(Fucα1-3)GlcNAc (Lewis x, Lex), GalNAcβ1-4(Fucα1-3)GlcNAc(LexNAc), and Galβ1-4(Fucα1-3)Glc, respectively. All human Fuc-Ts areknown to use Galβ1-4GlcNAc-type acceptors (7,9). GalNAcβ1-4GlcNAc servesalso as an acceptor for the Fuc-Ts of human milk (10). Human Fuc-Ts IIIand V have also α1-4fucosyltransferase activity using acceptors such asGalβ1-3GlcNAc (type I N-acetyl-lactosamine) to synthesizeGalβ1-3(Fucα1-4)GlcNAc (Lewis a). At least human Fuc-Ts III and V and(weakly VI) are able to fucosylate lactose and related oligosaccharidesto structures containing Galβ1-4(Fucα1-3)Glc sequences.

Enzymatic α1-3fucosylation of N-acetyl-chitobiose has been described, seU.S. Pat. No. 5,759,823, but the product was not characterized. Thereducing N-acetyl-chitobiose contains also the epimer in which there isManNAc at the reducing end, and it is not known from the data of thesaid U.S. patent if N-acetyl-chitobiose or its reducing-end epimer wasfucosylated. Saccharides with fucosylated reducing-end GlcNAc are notconsidered useful as they are labile and degrade even in aqueoussolutions at near neutral pH. The reducing-end fucosylated GlcNAc isvery rate, or non-existent, in mammalian natural oligosaccharides,possibly because of the lability which could make it useless also invivo for biological functions.

The present invention describes saccharide epitope analogues of themammalian fucosylated saccharide chains, as well as their synthesis. Aneffective method to synthesize such epitopes is to use α1-3fucosyltransferases or α1-3/4fucosyltransferases for fucosylation ofnovel acceptor sequences. Some of the acceptor sequences can besynthesized from cheap natural polysaccharides such as cellulose,chitin, chondroitin/chondroitin sulphates, or hyaluronic acid, alsonatural polysaccharides with the sequence Glcβ1-(3Glcβ1-4Glcβ1-)_(□)3Glccould be used to synthesize acceptors. β1-4GlcNAc transferase andUDP-GlcNAc can be used to generate GlcNAcβ1-4GlcNAcβ1- linked to Gal,GlcNAc or Man (11) and these can be used to make other analogues.Certain parasites have also been reported to containN-acetyl-chitooligosaccharides which could be used as acceptors for thefucosylation reaction (12).

Interestingly the novel fucosylations of N-acetyl-chitooligosaccharides(N-acetyl-chitotriose and larger) described here occured to thenon-reducing subterminal residue (forming a terminal Lewis x-likestructure with a linkage structure similar to human glycans) and not tothe reducing-end GlcNAc as in plant N-glycans.

SUMMARY OF THE INVENTION

In a first aspect, the present invention is directed to novelfucosylated oligosaccharides and oligosaccharide type compounds,especially N-acetyl-chitooligosaccharides which are α1-3 fucosylated inthe monosaccharide at the position subterminal to the non-reducing endof the oligosaccharide. The present invention is also directed to aprocess for the preparation of such compounds, which results in asite-specific fucosylation by the use of α1-3fucosyltransferase orα1-3/4fucosyltransferase enzyme to glycosylate the oligosaccharideacceptor substrate with L-fucose.

DESCRIPTION OF THE DRAWINGS

In the drawing,

FIG. 1(A) is a HPAE-chromatography of the neutral Fuc-TV fucosylationproducts of N-acetyl-chitotetraose. Fucosylated product (Glycan 4, FGN₄;for structures of Glycans 3-7, see Table 1) eluted at 7.62 min and theputative acceptor at 9.74 min. in the isocratic run with 40 mM NaOH. (B)shows HPAE-chromatographic purification of fucosylatedN-acetyl-chitohexaose (FGN₆) eluting at 7.09 min., putativeN-acetyl-chitohexaose peak at 9.04 min in the isocratic run with 40 mMNaOH. The saccharides were from Fuc-TV reaction.

FIG. 2 shows MALDI-TOF mass spectra of the key reaction mixtures andpurified product saccharides. F is L-Fucose, GN isN-acetyl-D-glucoseamine. (A) shows a mixture of FGN₄ produced by humanFuc-TV, (B) shows a mixture of FGN₆ and GN₆ produced by Fuc-TV. (C)shows FGN₆ purified by HPAE-chromatography. (D) showsexo-β-N-acetylhexosaminidase and novel endo-chitinase (the jack beanenzyme, mild conditions) reaction to the mixture of FGN₆ and GN₆produced by Fuc-TV. The major product is surprisingly FGN₅. (E) showsmild cleavage of the purified FGN₆ to mostly FGN₅ by the jack beanpreparation.(F) shows mild treatment of the purified FGN₄ by the jackbean preparation, 91% of the substrate remains intact.

FIG. 3. shows MS/MS spectra of reduced and permethylated fucosylatedN-acetyl-chitotriose 3 (A), and N-acetyl-chitotetraose 4 (B). Thefragment ion nomenclature of Domon and Costello is used to denote thegenerated fragments.

DETAILED DESCRIPTION OF THE INVENTION

Specifically the present invention concerns fucosylated oligosaccharidecompounds having the formula

In the formula I, A is H or a glycosidically β1-3 linkedD-glucopyranosyl residue (Glcβ1-3), R₁ is OH, R₂ is H and R₃ is OH oracylamido, —NH-acyl (i.e. monosaccharide 1 is Glc, or GlcNAcyl) or R₁ isH, R₂ is OH and R₃ is acetamido —NHCOCH₃ (i.e. monosaccharide 1 isGlcNAc), B is H, or an α-L-fucosyl or an α-L-fucosyl analogue, and R₄ isOH or acetamido —NHCOCH₃ (i.e. monosaccharide 2 is optionallyfucosylated Glc or GlcNAc), the curved line between the saccharide unitsindicating that the monosaccharide 1 is β1-4 linked to monosaccharide 2when B is linked to the position 3 of the monosaccharide 2, and themonosaccharide 1 is β1-3 linked to monosaccharide 2 when B is linked tothe position 4 of the monosaccharide 2, monosaccharide 1 is GlcNAc onlywhen monosaccharide 2 is Glc, n is 1 to 100, with the proviso that thereis always at least one α-fucosyl or α-fucosyl analogous group present inthe molecule, and

i) p and k are 0 and m is 1, in which case X is H, an aglycon residue ora monosaccharide selected from the group consisting of Glc, GlcNAc, Galor GlcNAc, optionally in reduced form, or oligosaccharide containing oneor more of said monosaccharide units, the monosaccharide 2 being β1-2,β1-3, β1-4 or β1-6 linked to saccharide X, with the proviso that X isnot H when both monosaccharides 1 and 2 are GlcNAc, B is L-fucosyl and nis 1, or

ii) p is 1, k is 0 or 1 and 1≦m≦1000, in which case X is a straightbond, or a mono- or oligosaccharide as defined under i),

Y is a spacer or linking group capable of linking the saccharide 2 or Xto Z, and Z is a mono- or polyvalent carrier molecule.

In the above formula, as well as below, Glc means a D-glucose residueand Gal means a D-galactose residue. Fuc or F means a L-fucose residue.GlcNAc or GN means a N-acetyl-D-glucose amine residue. Themonosaccharides are in pyranose form when glycosidically linked.

B as an analogue to the L-fucosyl residue is preferably a compound thatcontains a hydroxy-methyl group in place of the methyl group in 6position of fucosyl, that is L-galactosyl, or a deoxy derivative ofL-fucosyl, or an analogue where a di- to tetrasaccharide is linked toC6. Most preferably, however, B is H or L-fucosyl.

R₃ as an acylamido group is preferably an alkanoylamido group with 2 to24 carbon atoms and 0 to 3 double bonds between carbon atoms in astraight chain. Preferably R₃ is acetamido —NH—COCH₃, or analkanoylamido group with 8 to 24 carbon atoms, and 1 to 3 double bonds.m is preferably 1 to 100 and most preferably 1 to 10, and n ispreferably 1 to 10.

An oligosaccharide in the meaning of X contains preferably from 2 to 10monosaccharide units, the monosaccharide units preferably beingglycosidically β1-4 or β1-3 linked Glc or GlcNAc residues.

An aglycon group is preferably a hydrocarbon group, such as a C₁₋₂₀alkyl or C₂₋₂₀ alkenyl group, a C₃₋₁₀ cycloalkyl or cycloalkenyl group,or aryl or aralkyl group containing up to 10 carbon atoms in thearomatic ring, alkyl having the meaning given above, for example aphenyl group or benzyl group, or a heterocyclic group, that is acycloalkyl or an aryl group as defined containing one or moreheteroatoms O, S or N in the ring(s).

A preferred aglycon group is a lower alkyl or alkenyl group of 1 to 7,or 2 to 7 carbon atoms, respectively, or a phenyl or benzyl group. Apreferred heterocyclic aglycon group is 4-methylumbelliferyl.

The spacer group Y, if present, can be any group that is capable oflinking the group X or saccharide 2 to the carrier molecule Z, and suchgroups and methods of linking are known in the art, and alsocommercially available. For example when X is a saccharide, a bondbetween X and Y can be formed by reacting an aldehyde or a carboxylicacid with X at its C₁ or by introducing any aldehyde or carboxylic acidgroup in X through oxidation, to form a suitable bond such as —NH—,—N(R)— where R is an alkyl group, or a hydroxyalkylamine, an amide, anester, a thioester or thioamide. A suitable bond is also —O— or —S—, seee.g. Stowell et al Advances in Carbohydrate Chemistry and Biochemistry,37 (1980), 225-.

Thus the present invention provides fucosylated oligosaccharidecompounds as such or covalently bound to a carrier molecule. The carriermolecule can be mono- or polyvalent, and is preferably selected frompolymers, such as polyacrylamides, polyols, polysaccharides, such asagarose, bimolecules, including peptides and proteins, bovine or humanserum albumin being commonly used carriers for example in immunoassays.

A preferred group of compounds with the formula I comprises thefollowing

wherein the symbols have the meanings given in connection with theformula I above. In the above formula, the monosaccharides 1 and 2 arepreferably independently Glc and GlcNAc, B is L-fucosyl, and X is Glc orGlcNAc or a β1-3 or β1-4 linked oligomer comprising up to 10 units ofGlc and/or GlcNAc. When p and k=0 and m=1, the compounds have theformula

wherein the symbols have the same meanings as given above in the formulaIA, or X can also be H provided the monosaccharides 1 and 2 are bothGlc.

The compounds of the formula IA and IB are thus oligosaccharides thatare fucosylated in the subterminal or penultimate non-reducing endmonosaccharide. According to the invention it has been discovered thatthis specific fucosylation results in highly stable oligosaccharides,especially N-acetyl-chitooligosaccharides. According to a furtherpreferred embodiment of the invention, the saccharides have the formulaGlc/GlcNAcβ1-4(Fucβ1-3)Glc/GlcNAc(β1-4Glc/GlcNAc)_(n′)

In the above formula, n′ is the integer 1 to 8, preferably 1 to 6.

According to another embodiment of the invention, the saccharides havethe formulaGlcNAcylβ1-4(Fucα1-3)GlcNAc(β1-4GlcNAc)_(n′)wherein n′ has the meaning give above and acyl is an alkanoyl groupwhich preferably contains 8 to 24 carbon atoms and 1 to 3 double bonds.Preferably 1<n′<6, more preferably 2<n′<4.

According to the invention the compounds of the formula I are preparedby fucosylating a compound of the formula I wherein B is always H, witha donor nucleotide sugar containing L-fucose, or an analogue thereof, inthe presence of a fucosyltransferase enzyme, and optionally recoveringthe fucosylated saccharide so prepared. Such a reaction may be carriedout on the starting oligosaccharide prior to the optional binding theoligosaccharide to the carrier molecule Y. In the alternative, it isalso possible to fucosylate a carrier bound oligosaccharide. Such areaction is carried out essentially in the same manner as with non-boundoligosaccharides. For example, a N-acetyl-chitooligosaccharide such as aN-acetyl-chitotriose, can be linked to bovine serum albumin BSA througha spacer or it can be reductively aminated in a known manner to BSA, thereduced residue forming the spacer. In the fucosylation reaction,preferably an excess GDP-Fuc and a lower concentration of acceptor sites(0.1-1 mM) are used. If BSA is the carrier, the reaction buffer containsno non-glycosylated BSA. The products can be purified by methods ofprotein chemistry and the level of fucosylation can be checked byMALDI-TOF mass spectrometry, as described in more detail below. Thefucosylation can be repeated if the reaction is incomplete.

Oligosaccharides with an aglycon group as X can be fucosylatedessentially as has been described, preferably using lower acceptorconcentrations (0.1-1 mM). The products can be purified usingchromatographic methods including gel filtration and with partialreaction cleavage with N-acetylhexosaminidase from jack beans. As anexample, one obtains GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-benzyl fromGlcNAcβ1-4GlcNAcβ1-benzyl, GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-O-methyl fromGlcNAcβ1-4GlcNAcβ1-O-methyl, andGlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAcβ1-O-4-methylumbelliferyl fromGlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-O-4-methylumbelliferyl.

According to a preferred embodiment of the invention, thefucosyltransferase is human α1-3-fucosyltransferase or humanα1-3/4fucosyltransferase, especially one of human fucosyltransferasesIII-VII, IX, or α1-3/4fucosyl transferase of human milk. According toanother preferred embodiment, the L-fucose is in the form ofGDP-L-fucose.

According to an embodiment for making the preferred compounds describedabove, the starting oligosaccharide contains 3 to 10, especially 3 to 8β1-4 saccharide units. Such units are preferably N-acetyl-D-glucosamineresidues, GlcNAc, the corresponding starting materials thus beingN-acetyl-chitooligomers with formula (GlcNAcβ1-4)₃₋₁₀₍₈₎. According to asecond preferred embodiment, the oligosaccharides contain 2 to 10,especially 2 to 6 β1-4 D-glucose units, Glc, the corresponding startingmaterials thus being cellooligomers with formula (Glcβ1-4)₂₋₁₀₍₈₎.

According to the invention it is also possible to include a further stepof reacting the protect obtained with a β-N-acetyl-hexosaminidase undersufficiently strong conditions and in an amount sufficient to releasethe non-reducing terminal monosaccharide. It is also possible to reactthe product obtained with a β-N-acetyl-hexosaminidase under less severeconditions in order to release a monosaccharide from its reducingterminal. In this manner, a product is obtained which contains onesaccharide unit less than the primary oligosaccharide product. Thislatter reaction may also be controlled in such a manner that the enzymeprimarily degrades any remaining non-fucosylated substrate. This methodthus provides a convenient way of purifying the reaction mixture afterthe fucosylating step.

Due to the fucose unit, the novel oligosaccharides are stable compoundsand as such useful in a number of applications. As such they can, forexample, be used as substrates for testing, identifying anddifferentiating enzymes, such as chitinases, and when bound to a supportthey find use in immunoassays and affinity chromatography. They can alsofind use in a agrobiology, as stable plant protectants, as activators ofthe defence mechanisms and growth regulators of the plant cell similarlyas has been described with N-acetyl-chitooligosaccharides and their acylderivatives (13). In such use, incorporation of the fucose group in theoligosaccharide will in practice protect the oligosaccharide fromenzymatic degradation.

The α1-3/4fucosylated analogues of animal oligosaccharides are usefulfor studies of biological interactions involving their naturalcounterparts. The natural α1-3/4fucosylated oligosaccharides are knownto be ligands or counterreceptor of lectins mediating cell adhesion andother intercellular interactions. These saccharides are also importantantigenic epitopes recognized by anti-cancer or allergy-relatedantibodies. Some of these interactions are of special medical interestsuch as the leukocyte adhesion to blood vessels mediated by the bindingof the selectin proteins to their α1-3/4fucosylated counterreceptoroligosaccharides linked to proteins or lipids. The free oligosaccharideanalogues can be tested in in vitro or in vivo assays to find out theirabilities to inhibit the binding between the lectins and theircounterreceptors or alternatively the direct binding of theoligosaccharides to the lectins can be measured for example by affinitychromatography. The data obtained with the analogues in comparison tofree saccharide epitopes identical to the natural counterreceptoroligosaccharides reveal part of the specificity of the interaction. Itshows which modifications in the molecule are useful and which are nottolerated when better medical lead compounds for antagonists of theinteraction are designed. In search of better medical derivatives of theoligosaccharides the reducing end of the oligosaccharide chain can bemodified by numerous non-carbohydrate structures, aglycons. Freeoligosaccharides can also be useful as mixture of known composition (socalled libraries) in the tests of biological activities. Mixtures ofpositional fucosyl isomers are easily obtained by incubatingoligosaccharide with multiple acceptor sites with less GDP-Fuc than theamount of the acceptor sites or by following the reaction level byMALDI-TOF analysis of the reaction mixture and limiting the reactiontime (14).

Multi- or polyvalent conjugates of the oligosaccharides can also be moreactive antagonists of biological interactions when they are of naturaland non-antigenic type. Antigenic polyvalent conjugates such asoligosaccharides coupled to bovine serum albumin or to keyhole limpethemocyanin can be used to raise antibodies against the saccharideepitopes. Polyvalent conjugates conjugated to solid supports, such asagarose affinity chromatography media or plastic micro titer plates, arealso useful for assaying and purification of lectins and other proteinsbinding the saccharides. Fucosylated N-acetyl-chitooligosaccharideepitopes —Manβ1-4GlcNAc(Fucαa1-3)GlcNAcβ1-Asm of plant and insectproteins (15) are potent and cross relative human allergens andGlcNAc(Fucα1-3)GlcNAcβ1-conjugates can be useful for assaying allergyantibodies recognizing the epitope.

The selection proteins mediating vascular leukocyte adhesions are knownto bind sialyl-Lewis x [sLex, NeuNAcaα2-3Galβ1-4(Fucα1-3)GlcNAc],especially in sLexβ1-3Lexβ1-(16), and sialyl-Lewis a[NeuNAcα2-3Galβ1-3(Fucα1-4)GlcNAc] oligosaccharides (17) and in somereports also Lewis x [Lex, Galβ1-4(Fucα1-3)GlcNAc] (18) or VIM-2epitopes [NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc] (19).GalNAc-analogue of Lewis x, GalNAcβ1-4(Fucα1-3)GlcNAc has been reportedto be better selectin ligand than the sialyl-Lewis x (20, 21).

The following examples are intended to illustrate the invention.

The products according to the invention have been characterized bydegradation, mass spectrometry and NMR-experiments. The latter suggestthat the non-reducing end monosaccharide and the fucose are stacked in asolution conformation reminiscent of that dominating in Lex and LexNAcdeterminants. This lends significant protection to the fucosylatedoligosaccharides.

In the examples the following abbreviations have been used:

DQFCOSY, double quantum filtered correlation spectroscopy; ESI-CID,electrospray ionization-collision induced decay; Fuc & F, L-fucose;Fuc-Ts III-VIII and IX, humanα1-3fucosyltransferases/α1-3/4fucosyltransferases III-VII and IX;Fuc-Thm, α1-3/α1-3/4fucosyltransferases of human milk; Gal, D-galactose;GalNAc, D-N-acetylgalacosamine; GlcNAc & GN, D-N-acetylglucosamine;HPAEC-PAD, high pH anion exchange chromatography—pulsed amperometricdetection; Lac-NAc, Galβ1-4GlcNAc; LacdiNAc, GalNAcβ1-4GlcNAc, MALDI-TOFMS, matrix-assisted laser desorption/ionization time of flight massspectrometry; m/z, mass to charge ratio; MOPS,3-[N-Morpholino]propanesulphonic acid; ROESY, rotating frame nuclearOverhauser spectroscopy; ROE, rotating frame nuclear Overhauser; TOCSY,total correlated spectroscopy

In the examples, N-acetyl-chitotriose, N-acetyl-chitotetraose, andN-acetyl-chitohexaose were from Seikagau (Tokyo, Japan). Cellobiose wasfrom Thomas Kerfoot and Co.Ltd., and β-N-acetylhexosaminidase from jackbeans was from Sigma (St. Louis, Mo. U.S.A.). GDP-fucose (used in humanmilk experiments) was a kind gift from Prof. B. Ernst (UniversitätBasel, Switzerland). Human fucosyltransferases V and VI, recombinantproteins expressed in Spodeptera frugiperda, and GDP-fucose were fromCalbiochem (La Jolla, Calif., U.S.A.). GDP-[U-¹⁴C]fucose was fromAmersham International (Buckinghamshire, England). Superdex Peptide HR10/30 HPLC-column was from Pharmacia (Uppsala, Sweden). Dowex AG 1-X8(AcO⁻, 200-400 mesh) and Dowex AG 50W-X8 (H+, 200-400 mesh) and BiogelP-2 were from Bio-Rad (Richmond, Calif.). D₂O was from Cambridge IsotopeLaboratories (Woburn, Mass.). Partially purified human milkfucosyltransferases were prepared as described in (22) and assayed asdescribed in (23), 1 mU corresponds to transfer of 1 nmol of fucose to190 mM lactose/minute at 37° C., pH 7.5.

Fucosyltransferase reactions:

Fucosylation of N-acetyl-chitooligosaccharides with human milkfucosyltransferases (EC 2.4.1.152 and EC 2.4.1.65) was carried outessentially as described in (24), but with 2*360 μU of the enzyme(adding half of the enzyme after 2 days)/100 μl of reaction mixture andthe acceptor concentrations were 5 mM and by incubating reactions at 37°C. for four days. Fucosyltransferase V (Fuc-TV, EC 2.4.1.152,recombinant, Calbiochem) reactions were carried out under similarconditions but with 12.5 mU of the enzyme/100 μl, and the reactionmixtures were incubated at room temperature for five days. Vast excessesof GDP-Fuc in comparison to expected amount of products were used.Fucosyltransferase VI (EC 2.4.1.152, recombinant, Calbiochem) reactionswere carried out under the same reaction conditions as with Fuc-TVexcept 2 mM acceptor and 4 mM GDP-Fucose concentration and 10 mU of theenzyme/100 μl, and incubation for 3 days at 37° C. The reactions wereterminated by boiling for 3 minutes, the reaction mixtures were storedfrozen until analysed.

β-N-acetylhexosaminidase reactions.

Reactions catalyzed by jack bean β-N-acetylhexosaminidase (EC 3.2.1.30)under mild conditions were performed as described (25), using 0.3 mU ofthe enzyme/nmol releasable GlcNAc or in endochitinase reactions withFuc₁GN_(4,6) 6 nmol of saccharides were incubated 11 h with 38 mU of theenzyme.

Reactions performed under exhaustive reactions contained 300 mU of theβ-N-acetylhexosaminidase in 4.8 μl of 2.5 M (NH₄)₂SO₄, 2 nmol offucosylated N-acetyl-chito-oligosaccharides, and 40.2 μl of 50 mM sodiumcitrate pH 4.0. The reactions were incubated at 37° C., 300 mU of freshenzyme in 2.5 M (NH₄)₂SO₄ was added on days 2,3,5 and 7, and thereactions were stopped at day 8 by adding 1 vol of ice-cold ethanol and8 volumes of ice-cold water.

Reduction of oligosaccharides.

Oligosaccharides were reduced with NaBH₄ essentially as described in(26). The alditols were purified by gel filtration and the completenessof the reactions was verified by MALDI-TOF mass spectrometry.

Chromatographic methods.

Samples from the enzymatic reactions were desalted by passing them inwater through 1.5 ml of Dowex AG-50 (H+, 200-400 mesh) and 1.5 ml ofDowex AG-1 (AcO-, 200-400 mesh) and then purified by gel filtration HPLCin a column of Superdex Peptide HR 10/30, with ultra pure water or 50 mMNH₄HCO₃ as eluant, at a flow rate of 1 ml/min. Gel filtration in aBiogel P-2 columns (1×142 cm) was performed with ultrapure water,UV-absorbance was monitored at 214 nm. High-pH anion exchangechromatography with pulsed amperometric detection (HPAEC-PAD) wasperformed on a (4×250 nm) Dionex CarboPac PA-1 column (Dionex, CA), thesamples were run isocratically with 40 or 60 mM NaOH.

Mass spectrometry.

Matrix-associated laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS) was performed in the positive ion delayedextraction mode with a BIFLEX™ mass spectrometer (Bruker-FranzenAnalytik Bremen, Germany) using 2,5-dihydroxybenzoic acid as the matrix.The matrix peaks at the low mass region were “suppressed” by using highsample concentrations (100 pmol/μl).

Electrospray mass spectra (ESI-MS) were collected using an API365 triplequadrupole mass spectrometer (Perkin-Elmer instruments, Thornhill,Ontario, Canada). Samples were dissolved in 50% aqueous methanolcontaining 0.5 mM sodium hydroxide, and injected into the massspectrometer with a nanoelectrospray ion source (Protana A/S, Odense,Denmark) at a flow rate of about 30 nl/min. MS/MS spectra were acquiredby colliding the selected precursor ions to nitrogen collision gas withacceleration voltages of 35 V (doubly-charged precursors) or 55 V(singly charged precursors).

NMR spectroscopy.

Prior to NMR experiments the saccharides were lyophilized twice from D₂Owith then dissolved in 300 μL of D₂O (99.996 atom %, Cambridge IsotopeLaboratories, Woburn, Mass., U.S.A.). The NMR experiments were carriedout on a Varian Unity 500 spectrometer at 23° C. using Shigemi tubes(Shigemi Co., Tokyo, Japan). In recording ID proton spectra amodification of the WEFT sequence (27) was used. For the DQFCOSY (28)and TOCSY (29) experiments (32 scans per t₁ value) matrices of 2k*256and 4k*256 points were collected, and zero-filled to 2k*512 and 4k*512points, respectively. A 90° shifted sine-bell weighting function wasemployed in both dimensions. In TOCSY, a spin-lock time of 100 ns(MLEV-17) was used. In order to resolve overlap within spin systems 1Dselective TOCSY (30) spectra were recorded with mixing times varyingfrom 10 ms to 140 ms and a gaussian selective pulse. In recording theROESY spectrum (31, 32) the transmitter was placed outside the signalarea at 5.750 ppm and a continuous-wave spin-lock with spin-lock time of300 ms was employed. A matrix of 2k*256 was collected and zero-filled to2k*512 points. A 90° shifted sine-bell weighting function was used inboth dimensions. Additionally, the t₁ time domain data was doubled usingforward-backward linear prediction.

For the DEPT(135) (33) spectrum 92,000 points were recorded with aspectral width of 18,000 Hz. For the HSQC (34) and 2D HSQC-TOCSY (35)(48 and 56 scans per t₁ value, respectively), matrices of 2k*128 and2k*256 points were recorded and zero-filled to 2k*256 and 2k*512 points,respectively and a shifted sine-bell function was used. In the 2DHSQC-TOCSY a mixing time of 120 ms was employed. The ¹H and ¹³C chemicalshifts were referenced to internal acetone, 2.225 and 31.55 ppm,respectively.

EXAMPLE 1

Human Fuc-TV-catalyzed reaction of N-acetyl-chitotriose and GDP-Fucose

The oligosaccharide mixture, generated by incubation ofN-acetyl-chitotriose (1000 nmol, 5 mM) with GDP-Fuc (1000 nmol, 5 mM)and 25 mU Fuc-TV at room temperature for 5 days was desalted andisolated by gel filtration HPLC. MALDI-TOF-MS revealed that the mixturecontained 32 mol % fucosylated N-acetyl-chittotriose and 68%N-acetyl-chitotriose. This mixture was subjected to the “mild treatment”with jack bean β-N-acetylhexosaminidase, which cleaved most of thesurplus N-acetyl-chitotriose substrate but only a small amount of thefucosylated N-acetyl-chitotriose product. A representative sample of alloligosaccharides was isolated from a 5% aliquot of the digest by usinggel filtration HPLC. MALDI-TOF MS analysis of showed that it consistedof fucosylated N-acetyl-chitotriose 67 mol %, N-acetyl-chitotriose 7%,Fuc₁GlcNAc₂ 12%, N-acetyl-chitobiose 13%.

The monofucosylated N-acetyl-chitotriose was purified from the rest ofthe β-N-acetylhexosaminidase digest by gel filtration HPLC. MALDI-TOF-MSanalysis of the purified Fuc₁GlcNAc₃, Glycan 3, revealed the presence ofonly a 5% N-acetyl-chitotriose contamination. The total yield of thepurified Glycan 3 was 191 nmol (19%).

Mass spectrometric characterization of Fuc-TV generated Glycan 3

An aliquot of the monofucosylated N-acetyl-chitotriose was reduced andpermethylated and subjected to ESI-MS. The doubly-charged [M+2Na]²⁺ ion(m/z 508.8) was first selected for MS/MS (FIG. 3A). The fragmentsobtained could all be assigned to a tetrasaccharide carrying the fucoseunit in the middle GlcNAc units, i.e.GlcNAcβ1-(Fucα1-)GlcNAcβ1-GlcNAcol. A loss of terminal, nonsubstitutedGlcNAc unit is evident from the B₁ ions at m/z 282.2 (sodiated) and m/z260.0 (protonated). Loss of methanol from the m/z 260.0 ion accounts forthe m/z 228.2 and m/z 196.2 ions. The Y₁ ions at m/z 316.2 and m/z 338.2(carrying one and two sodiums, respectively) indicate that the GlcNAcalditol carried only one monosaccharide substitutent. Furthermore, theY_(2α)/B₂ ion at m/z 442.4 can only arise by loss of terminalunsubstituted GlcNAc and the reduced GlcNAc residue. No fragments wereobserved even in closer inspection, which would represent fucosylatedreduced end (i.e. Fucα1-GlcNAcol, m/z 490.4) or fucosylated nonreducingend GlcNAc (terminal Fucα1-GlcNAc, m/z 456.2). The origin of the fairlyintense fragment at m/z 455.2 is somewhat complex. Its nature wasrevealed by producing the B₂, Y_(2α) and Y_(2β)-H2O fragments with ahigh orifice voltage, and by collecting MS/MS/MS data with these skimmerfragments (not shown). Only the Y ions generated the m/z 455 ion, so itmust contain the reducing end. We suggest that this is an ^(0,4)X₁ ion,arising by a cross-ring cleavage of the penultimate GlcNAc ring.

NMR-spectroscopy of Fuc-TV generated Glycan 3

The data from 1D 1H-NMR-spectrum of Fuc-TV-generated Glycan 3 isreported in Table 2. Table 1 indicates the number of the residues usedin Table 2. The H1, H5 and H6 signals of fucose in Glycan 3 resembletheir counterparts in GlcNAcβ1-4(Fucα1-3)GlcNAc glycans (36) and inGlycan 4 analyzed by 2D NMR (Table 2), but are distinct from those in—GlcNAcβ1-4(Fucα1-6)GlcNAc saccharides (37). Taken together, theNMR-data confirm that the fucosylated N-acetyl-chitotriose, Glycan 3,generated by Fuc-TV is GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAc.

The reaction of N-acetyl-chitotriose and GDP-Fucose catalyzed by humanmilk Fuc-Ts

Incubation of N-acetyl-chitotriose (1000 nmol, 5 mm) with GDP-fucose(600 nmol, 3 mM) and 1.4 mU of the enzyme for four days at 37° C. gavean oligosaccharide mixture. After ion exchange desalting, mildβ-N-acetylhexosaminidase treatment, and repeated gel filtration HPLCruns, a sample of 221 nmol of purified fucosylated N-acetyl-chitotriosewas obtained. MALDI-TOF MS revealed that the product contained 92% offucosylated N-acetyl-chitotriose and 7% of N-acetyl-chitotriose. The 1D1H-NMR-spectrum of the product was almost identical with that ofFuc-TV-generated Glycan 3. The ESI-MS-data of the Glycan 3 in reducedform, too, were identical to those obtained with reduced derivative ofthe Fuc-TV-generated Glycan 3 (not shown).

EXAMPLE 2

The Fuc-TV-catalyzed reaction of N-Acetyl-Chitotetraose and GDP-Fucose

Incubation of N-acetyl-chitotetraose (1000 nmol, 5 mM) with GDP-Fuc(1000 nmol, 5 mM) and 25 mU human Fuc-TV at room temperature for 5 days,and isolation of the resulting oligosaccharide mixture by using gelfiltration HPLC gave a product consisting of 32 mol % fucosylatedN-acetyl-chitotetraose and 68% of unreacted N-acetyl-chitotetraose (FIG.2A). The mild treatment of the mixture with jack beanβ-N-aceylhexosaminidase followed by chromatographic removal of theliberated from GlcNAc gave an oligosaccharide mixture consisting ofFuc₁GlcNAc₄ 50%, GlcNAc₄ 6%, Fuc₁GlcNAc₃ 8%, GlcNAc₃ 14%, GlcNAc₂ 22 mol%. The composition of this mixture implies that also the fucosylatedN-acetyl-chitotetraose was cleaved much slower thanN-acetyl-chitotetraose by the β-N-acetylhexosaminidase, confirming theanalogous data on degradation of Glycan 3. The oligosaccharide mixturewas then fractioned by gel filtration HPLC, yielding a purified product(225 nmol) that consisted of 93 mol % of fucosylatedN-acetyl-chitotetraose and 7% N-acetyl-chitotetraose.

Another fucosylation mixture consisting of 11% of the fucosylatedN-acetyl-chitotetraose and 89% of N-acetyl-chitotetraose was separatedin an isocratic HPAE run in a (4×250 mm) Dionex CarboPac PA-1 column(Dionex, Sunnyvale, Calif.) column by using with 40 mM NaOH as theeluent (FIG. 1A). The peak eluting at 7.62 min contained the fucosylatedN-acetyl-chitotetraose. The 1D 1H-NMR spectrum of the purified product,Glycan 4 synthesized by Fuc-TV was almost identical with that of Glycan4 synthesized by the Fuc-Ts of human milk (see below).

A sample of the purified Glycan 4 was degraded by the exhaustivetreatment with jack bean β-N-acetylhexosaminidase, yielding anoligosaccharide that behaved in gel filtration as a compound about 1.5glucose unit smaller then N-acetyl-chitotetraose, suggesting that it hadlost a GlcNAc unit. This view was confirmed by applying MALDI-TOF-MSanalysis, which revealed 89 mol % Fuc₁GlcNAc₃ and 11 mol % Fuc₁GlcNAc₂.The minor product species had probably lost both the non-reducing endGlcNAc and the reducing end GlcNAc during the exhaustive cleavage (seebelow, at the mild jack bean β-N-acetylhexosaminidase treatment of thefucosylated N-acetyl-chitohexaose).

Put together, the analytical data of Fuc-TV generated Glycan 4 areconsistent with the structure ofGlcNAcβ1-4(Fucα1-3(GlcNAcβ1-4GlcNAcβ1-4GlcNAc.

Glycan 4-synthesis catalyzed by human milk Fuc-Ts

N-Acetyl-chitotetraose (6.0 μmol, 5 mM), and GDP-Fuc (3.0 μmol, 2.5 mM)were incubated with 8.7 mU of the enzyme for four days at 37° C. Afterdesalting and gel filtration HPLC-run, the fucosylated product wasseparated from the non-fucosylated N-acetyl-chitotetraose by HPAEC usingisocratic run with 40 mM NaOH (not shown). The purified product wasdesalted by ion exchange and was further purified by gel filtration HPLCyielding 744 nmol monofucosylated N-acetyl-chitotetraose. MALDI-TOF massspectrometry of the product revealed that it was uncontaminated byN-acetyl-chitotetraose acceptor (not shown).

A sample of the purified Glycan 4 generated by human milk Fuc-Ts wastreated exhaustively with jack bean β-N-acetylhexosaminidase.MALDI-TOF-MS analysis of the HPLC-purified oligosaccharides of thedigest revealed 91 mol % Fuc₁GlcNAc₃ and 9% Fuc₁GlcNAc₂.

Mass spectrometric characterization of Glycan 4 generated by human milkFuc-Ts

An aliquot of the monofucosylated N-acetyl-chitotetraose was reduced andpermethylated and subjected to ESI-MS. The doubly-charged [M+2Na]²⁺ ion(m/z 631.6) was selected for MS/MS (FIG. 3B). The low-mass region of theMS/MS spectrum resembles that of the monofucosylatedN-acetyl-chitotriose, showing the same B₁ fragment s for theunsubstituted terminal GlcNAc unit (m/z 282.2, m/z 260.2), and Y₁ ionsfor the reducing end GlcNAc alditol (m/z 316.2, m/z 338.2).

In addition, the Y_(2α)/B₂ ion (m/z 442.4), the B₃ ion (m/z 946.8), andthe Y_(3α) ion (m/z 980.6) verify that the fucose unit is linked toeither of the midchain GlcNAc units. The Y₂ ion at m/z 561.4 indicatesthat this disaccharide fragment only carried one monosaccharidesubstituent, and thus the fucose must reside at the penultimate GlcNAcresidue. This is directly confirmed by the B₂ ion at m/z 701.6, carryingthe non-reducing terminus structure GlcNAcβ1-(Fucα1-) GlcNAc. Evencloser investigation of the minute fragments in the MS/MS data did notreveal any specific fragments for other pentasaccharide isomers, and wetherefore conclude that N-acetyl-chitotetraose is fucosylated solely tothe penultimate GlcNAc residue next to the non-reducing end.

NMR-spectroscopic analysis of Glycan 4 produced by human milk Fuc-Ts

The 1D 1H-NMR spectrum of the purified product, Glycan 4 (Table 3)revealed fucose H1, H5 and H6 signals characteristic to α3-linked fucoserather than α6-linked fucose in unconjugated N-glycans (36, 37). The H4signal of the distal GlcNAc6 was similar to its analogs in Glycan 3,confirming that the fucose is linked to the penultimate GlcNAc unitclose to the non-reducing end. The proton signals of the fucose and thenon-reducing end GlcNAc6 were assigned from DQFCOSY and TOCSY spectra ofGlycan 4. The 2NMR-data strengthen the notion that the non-reducing endelements of Glycan 4 resemble those of Glycan 3.

Taken together, the NMR-data confirm the MS-results and the degradationdata, establishing that Glycan 4 generated by human milk Fuc-Tsrepresented also GlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAcβ1-4GlcNAc.

EXAMPLE 3

Human FucTV-Catalyzed Reaction of N-Acetyl-Chitohexaose and GDP-Fucose

N-Acetyl-chitohexaose (1.0 μmol, 5 mM) was fucosylated with GDP-Fucose(1.0 μmol, 5 mM) and 25 mU Fuc-TV at room temperature for 5 days.MALDI-TOF MS of the purified representative mixture of the productoligosaccharides showed that 31% of N-acetyl-chitohexaose wasfucosylated (FIG. 2B). The product saccharides were further purified inin HPAE-chromatography run isocratically with 40 mM NaOH (FIG. 1B). Thepeak eluting at 7.09 min was pooled, was desalted and was furtherpurified by gel filtration HPLC. The product heptasaccharide (195 nmol)showed in MALDI-TOF analysis 96% of monofucosylatedN-acetyl-chitohexaose, Glycan 6, and 4% of N-acetyl-chitohexaose, FIG.2C. The 1D 1H NMR spectrum (Table 2) shows the H1 signals in theGlcNAc5, the GlcNAc6 and the fucose units in Glycan 6, which are nearlyidentical with their counterparts in Glycan 4. This suggests that theGlycan 6 isGlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAc. Thisnotion is further supported by the identical H4-signals of the distalGlcNAc6 units and the fucose H5 resonances in the two glycans. Thesimilarity of this latter pair of signals in the two glycans isparticularly significant, because they are known to interact in Glycan 4(see above), and are likely to be very sensitive to structuraldifferences. Likewise, the similarity of fucose H5 signals in Glycan 6,and Glycan 4 speaks for the identity of the nonreducing area in thesethree saccharides.

Treatment of Glycan 6 with jack bean β-N-acetylhexosaminidase

A Fuc-TV-generated mixture of Glycan 6 and N-acetyl-chitohexaose (27:73mol/mol (FIG. 2B)) was subjected to the mild jack beanβ-N-acetylhexosaminidase treatment as described earlier. The resultingoligosaccharides were isolated as a representative mixture by using gelfiltration HPLC. MALDI-TOF MS revealed that mixture consisted ofFuc₁GlcNAc₆ (3.5 mol %), GlcNAc₆ (1.7%), Fuc₁GlcNAc₅ (40%), Fuc₁GlcNAc₄(3.5%); small N-acetyl-chitooligosaccharides ranging fromN-acetyl-chitobiose to N-acetyl-chitopentaose were also observed (FIG.2D). The principal product of the digest, the monofucosylatedN-acetyl-chitopentaose, Glycan 5, was purified in a gel filtrationHPLC-run. MALDI-TOF MS suggested that Glycan 5 was obtained hereby in apurity of 83%; it was contaminated by several penta- andhexasaccharides. The chemical shifts are presented in Table 2. The NMRdata show nearly identical H1 signals in the GlcNAc5, the GlcNAc6 andthe fucose units in Glycan 5 and Glycan 4, suggesting, that thestructure of the former isGlcNAcβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAc.

This notion is further supported by the identical H4-signals of thedistal GlcNAc6 units and the fucose H6 resonances in the two glycans.Hence, the mild jack bean β-N-acetylhexosaminidase treatment hassurprisingly released a GlcNAc unit from the reducing end of Glycan 6,but has not attacked the reducing end of the product, Glycan 5, asefficiently.

Treatments of purified Glycan 6 and Glycan 4 by the endo-chitinaseactivity of jack bean β-N-acetylhexosaminidase

When the purified Fuc-TV-generated Glycan 6 was subjected to the mildtreatment with jack bean β-N-acetylhexosaminidase, the resulting digestshowed in MALDI-TOF MS only 5 mol % of intact substrate; 85% of thematerial was converted into Fuc₁GlcNAc₅ and 11% into Fuc₁GlcNAc₄ (FIG.2E). The data confirms that the endochitinase cleaves fast Glycan 6,releasing mainly one GlcNAc unit from the reducing end and the enzymemay be able to cleave off also a N-acetyl-chitobiose unit from thereducing end of Glycan 6, generating Glycan 4. Alternatively, Glycan 4may have been formed by the release of a GlcNAc unit from the reducingend of Fuc₁GlcNAc₅.

When the purified Fuc-TV-generated Glycan 4 was subjected to the mildtreatment with jack bean β-N-acetylhexosaminidase, the resulting digestshowed in MALDI-TOF MS that most (91 mol %) of the substrate hadsurvived and only 6% of Fuc₁GlcNAc₃ had been formed; 3% of the materialwas found as fucose-free N-acetyl-chitotetraose (FIG. 2F). Hence, theendo-chitinase activity appeared to cleave off slowly one GlcNAc unitfrom the reducing end of Glycan 4.

EXAMPLE 4

Fucosylation of cellobiose

Cellobiose (4000 nmol, 20 mM, from Thomas Kerfoot and Co. ltd) wasincubated with GDP-[¹⁴C]Fuc (1000 nmol, 5 mM, 100 000 cpm) andrecombinant fucosyltransferase V (25 mU, 50 ml of the commercial enzymepreparation from Calbiochem) in 50 mM MOPS-NaOH pH 7.5 containing 8 mMMnCl₂, 1000 mM NaCl, 1 mg/ml BSA and 0.02% NaN₃ in total volume of 200ml at room temperature for 4 days and 16 hours. The reaction productswere desalted by running through 1.5 ml beds of Dowex AG 1-X8 and DowexAG 50W-X8. Part of the products were run in paper chromatography whichshowed a trisaccharide like product migrating close to maltriose(Rmaltotriose=0.97 marker, glycerol in the sample may affect themigration) and a broad peak (broadened by glycerol in the sample)probably corresponding to free [¹⁴C]Fuc. Solvent A was used as describedin (38), using the upper phase of n-butanol-acetic acid:water (4:1:5,v/v, solvent A) and scintillation counted from small strips of paper.Major part (75%) of the products were run in gel filtrationchromatography in Biogel P-2 (Biorad) column. A peak of radioactiveproducts (256 nmol by radioactivity, calculated total yield 340 nmol)eluting at position expected for monofucosylated cellobiose was obtainedand another radioactive peak containing [¹⁴C]Fuc-like material.Fractions in the first half of the product peak were analyzed to containsaccharide with size corresponding to fucosylated cellobiose inMALDI-TOF mass spectrometry, monoisotopic m/z [M+Na]+ close to 511.2 andm/z [M+K]+ close to 527.2, the fractions contained very minor amounts orno other possible saccharides like cellobiose or cellotriose. The pooledsaccharide (150 nmol by radioactivity) were analyzed byNMR-spectroscopy. NMR-spectroscopy revealed signals of Fuc H1α at 5.425ppm, Fuc H1β at 5.369 ppm and signals of the reducing Glc H1a at 5.189ppm and Glc H1β at 4.655 ppm similarly as described for—Galβ1-4(Fucα1-3)Glc (39).

A product (<0.5 pmol) migrating similarly in paper chromatography(Rmaltotriose=1.04) can be obtained by incubating lysate (40 mg ofprotein) of CHO-cells expressing full length Fuc-TV with GDP-[¹⁴4C]Fuc(1 nmol, 100 000 cpm) and 100 nmol of cellobiose for 1 hour as describedin (40).

EXAMPLE 5

Reactions with laminaribiose and laminaritetraose

Glcβ1-3Glc+GDP-Fuc→Glcβ1-3(Fucα1-4)Glc

Glcβ1-3Glcβ1-3Glcβ1-3Glc+GDP-Fuc→

Glcβ1-3(Fucα1-4)Glcβ1-3Glcβ1-3Glc

+Glcβ1-3Glcβ1-3(Fucα1-4)Glcβ1-3Glc

+Glcβ1-3Glcβ1-3Glcβ1-3(Fucα1-4)Glc

Laminaribiose (1000 nmol, 5 mM, Seikagaku) and laminaritetraose (1000nmol, 5 mM, Seikagaku) were reacted with GDP-[¹⁴C]Fuc (1000 nmol, 5 mM,100 000 cpm) and recombinant fucosyltransferase V (25 mU, 50 ml of thecommercial enzyme preparation from Calbiochem) as above. The products ofboth reactions were desalted as above. The products from laminaribiosewere purified by P-2 gel filtration chromatography and radioactivetrisaccharide-like product was obtained.

The desalted products from laminaritetraose were also purified by P-2gelfiltration chromatography and a radioactive (174 nmol byradioactivity) peak eluting a pentasaccharide product as expected, wasobtained, 131 nmol of product with monoisotopic m/z [M+Na]⁺ close(difference less than 0.1%) to 835.3 and m/z [M+K]⁺ close to 851.3 werepooled from front and middle fractions of the peak, the fractionscontained less than 4% of the acceptor, no other saccharide productswere observed in analysis by MALDI-TOF mass spectrometry. The purifiedproduct saccharides were analyzed by NMR spectroscopy.

EXAMPLE 6

Desulfation, reduction, and fucosylation of chondroitin sulfate

Chondroitin sulfate (shark cartilage, Sigma) was cleaved byhyaluronidase (bovine testes, Sigma; chondroitin sulfates can be alsocleaved with chondroitinases giving oligosaccharides with delta-uronicacid at non reducing end) to sulfated oligosaccharides suchas[GlcAβ1-3(sulf-6)GalNAcβ1-4]nGlcAβ1-3(sulf-6)GalNAc. Fractionscontaining tetrasaccharide-like materials were purified and desulfatedas essentially as described in (41). Tetrasaccharide and hexasaccharidefractions were purified by gel filtration and anion exhangechromatographies. Similar oligosaccharides were also produced by firstdesulfating and then cleaving by hyaluronidase and purifying theproducts. The GlcA-residues of the tetrasaccharide were reduced to Glcby EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NaBH₄. Thereduced tetrasaccharide Glcβ1-3GalNAcβ1-4Glcβ1-3GalNAcol was purifiedand fucosylated using GDP-Fuc and human milk fucosyltransferase(s). TheMALDI-TOF mass spectrometry revealed peaks at m/z 919.8 and m/z 935.8corresponding to the product Glcβ1-3GalNAcβ1-4(Fucα1-3)Glcβ1-3GalNAcol(calc. m/z [M+Na]⁺ is 919.7 and m/z [M+K]⁺ is 935.7).

EXAMPLE 7

GlcNAcylβ1-4GlcNAcβ1-4GlcNAcβ1-4GlcNAc, wherein Acyl istrans-9-octadecenoyl, synthesis of the acceptor being described in (13),can be incubated with Fuc-TVI and MnCl₂ under conditions described abovefor Fuc-TVI, but acceptor concentrations between 0.1-0.5 mM arepreferred. The product GlcNAcylβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAcβ1-4GlcNAcis purified chromatographically and analysed by NMR-spectroscopy andmass spectrometry.

EXAMPLE 8

N-Acetyl-chitotetraose can be incubated with human Fuc-TVI and MnCl₂under conditions described above for Fuc-TVI. The productGlcNAcβ1-4(Fucα1-3(GlcNAcβ1-4GlcNAcβ1-4GlcNAc can be obtained withalmost quantitative yield and purified by HPAE-chromatography. TheNMR-data and mass spectroscopy data of the product are practicallyidentical with the fucosylated N-acetyl-chitotetraose obtained byFuc-TV.

TABLE 1 Structures of the fucosylated N-acetyl-chito-oligosaccharides inpresent study. GN is GlcNAc and Fuc is L-fucose, line - indicatesβ1-4-linkage and line / is α1-3-linkage, residue numbering is italics. 6 5  4 6  5  4  3 3 GN-GN-GN 4 GN-GN-GN-GN Fuc/ Fuc/ 6  5  4  3  2 6  5 4  3  2  1 5 GN-GN-GN-GN-GN 6 GN-GN-GN-GN-GN-GN Fuc/ Fuc/ 6  5  4  3 7GN-GN-GN-GN

TABLE 2 ¹H Chemical shifts (ppm) of structural reporter groups forGlycans 3-7 at 23° C. Glycans Proton Residue 3 4 5 6 7 H1 1 — — —5.181(α)/4.688(β) — 2 — — 5.182(α)/4.688(β) 4.578/4.565* — 3 —5.184(α)/4.689(β) 4.579/4.567* 4.565 5.185(α)/4.689(β) 45.185(α)/4.688(β) 4.579/4.570* 4.567 4.565 4.58 5 4.580/4.571* 4.5704.567 4.565 4.58 6 4.522 4.519 4.517 4.516 4.58 fucose 5.124 5.119 5.1175.116 — H4 6 3.238 3.237 3.236 3.235 n.d. H5 fucose 4.764 4.762 4.7624.758 — CH3 fucose 1.268 1.267 1.265 1.264 — n.d. = not determined *Thetwo chemical shift values given arise from signals representing the α-and β-pyranosic forms of the oligosaccharides respectively.

TABLE 3 NMR-data of the Glycan 4. Proton GlcNAc6 Fucose H1 4.519 5.119H2 3.735 3.707 H3 3.535 3.938 H4 3.237 3.803 H5 3.428 4.762 H6 3.607 —H6′ 3.963 — CH3 — 1.267

REFERENCES

-   1. Johnston, D. S. Wright, W. W., Shaper, J. H., Hooke, C. H., Van    den Eijnden, D. H., and Joziasse, D. H. (1998) J. Biol. Chem.    273(4), 188-1895-   2. Gooi, H. C., Feizi, T., Kapadia, A., Knowles, B. B., Solter, D.,    and Evans, M. J. (1981) Nature 292, 156-158-   3. Dodd, J., and Jessel, T. M. (1986) J. Exp. Med. 124, 225-238-   4. Oudega, M., Marani, E., and Thomeer, R. T. W. M. (1992)    Histochem. J. 24, 869-877-   5. Butcher, E. C., and Picker, L. J. (1996) Science 272, 60-66-   6. McEver, R. P. (1997) Glycoconjugate J. 14, 585-591-   7. Natsuka, S. Gersten, K. M., Zenita, K., Kannagi, R., and    Lowe, J. B. (1994) J. Biol. Chem. 269, 16789-16794-   8. Edbrooke, M. R., Britten, C. J., Kelly, V. A. M., Martin, S. L.,    Smithers, N., Winder, A. J., Witham, S. J., and Bird, M. I. (1997)    Biochem. Soc. Transact. 25, 880-887-   9. Kaneko, M., Kudo, T., Iwasaki, H., Ikehara, Y., Nishihara, S.,    Nakagawa, S., Sasaki, K., Shiina, T., Inoko, H., Saitou, N., and    Narimatsu, H. (1999) FEBS Lett. 452, 237-242-   10. Bergwerff, A. A., van Kuik, J. A., Schiphorse, W. E. C. M.,    Koeleman, C. A. M., van den Eijnden, D. H., Kamerling, J. P., and    Vliegenthart, J. F. G. (1993) FEBS Lett. 334, 133-138-   11. Bakker, H., Scoebnakers, P. S., Koeleman, C. A. M., Joziasse, D.    H., van Die, I and van den Ejnden, D. H. (1997) Glycogiology 7,    539-548-   12. Haslam, S. M., Coles, G. C., Munn, E. A., Smith, T. S.,    Smith, H. F., Morris, H. R., and Dell, A., (1996) J. Biol. Chem.,    271, 30561-30570.-   13. Rohrig, H., Schmidt, J., Walden, R., Czaja, I., Miklasevics, E.,    Wieneke, U., Schell, J., and John M. (1995) Science 269, 841-843-   14. Niemelā, R., Natunen, J., Penttilā, L., Salminen, H., Helin, J.,    Maaheimo, H., Costello, C. E., and Renkonen, O. (1999) Glycobiology    9, 517-526-   15. Wilson, I. B. H., Harthill; J. E., Mullin, N. P., Ashford, D.    A., and Altman, F. (1998) Glycobiology 8, 651-661-   16. Wilkins, P. P., McEver, R. P., and Cummings, R. D. (1996) J.    Biol. Chem. 271, 1873-18742-   17. Varki, A. (1994) Proc. Natl. Acad. Sci. USA 91, 7390-7397-   18. Larsen, E., Palabrica, T., Sajer, S., Gilbert, G. E., Wagner, D.    D., Furie, B. C., and Furie, B. (1990) Cell 63, 467-474-   19. Stroud, M. R., Handa, K., Salyan, M. E. K., Ito, K., Levery, S.    B., Hakomori, S., Reinhold, B. B., and Reinhold, V. N. (1996)    Biochemistry 35, 770-778-   20. Jain, R. K., Piskorz, C. F., Huang, B.-G., Locke, R. D., Han,    H.-L., Koenig, A., Varki, A., and Matta, K. L. (1998) Glycogiology    8(7), 707-717-   21. Grinnell, B. W., Hermann, R. B., and Yan, S. B. (1994)    Glycobiology 4(2), 221-225-   22. Natunen, J., Niemelā, R., Penttilā, L., Seppo, A., Ruohtula, T.,    and Renkonen, O. (1994) Glycobiology 4, 577-583-   23. Eppenberger-Castori, S., Lōtscher, H., and Finne, J. (1989)    Glycoconjugate J. 6, 101-114-   24. Palcic, M. M., Vernot, A. P., Ratcliffe, R. M., and    Hindsgaul, O. (1989) Carbohydr. Res. 190, 1-11-   25. Renkonen, O. Helin, J., Penttilā, L., Maaheimo, H., Niemelā, R.,    Leppānen, A., Seppo, A., and H{dot over (a)}rd, K. (1991)    Glycoconjugate J. 8, 361-367-   26. Rasilo, M.-L., and Renkonen, O. (1982) Hoppe Syler's Z. Physiol.    Chem. 363, 89-93-   27. H{dot over (a)}rd, K., van Zadelhoff, G., Moonen, P.,    Kamerling, J. P., and Vliegenthart, J. F. G. (1992) Eur. J. Biochem.    209, 895-915-   28. Marion, D., and Wüthrich, K. (1985) Biochem. Biophys. Res.    Commun. 117, 967-974-   29. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 65, 355-360-   30. Xu, G. and Evans, J. S., (1966) J. Magn. Reson., Ser. B 111,    183-185-   31. Bothner-By, A. A., Stephens, R. L., Lee, J., Warren, C. D., and    Jeanloz, R. W. (1984) J. Am. Chem. Soc. 106, 811-813-   32. Bax, A., and Davis, D. G. (1985) J. Magn. Reson. 63, 207-213-   33. Doddrell, D. M., Pegg, D. T., and Bendall, M. R. (1982) J. Magn.    Reson. 48, 323-327-   34. Kay, L. E., Keifer, P., and Saarinen, T. (1992) J. Am. Chem.    Soc. 114, 10663-10665-   35. Wijmenga, S. S. (1989) J. Magn. Res. 84, 634-642-   36. Lhernould, S., Karamanos, Y., Bourgerie, S., Strecker, G.,    Julien, R., and Morvan, H. (1992) Glycoconjugate J. 9, 191-197-   37. Lawrence, C. W., Little, P. A., Little, B. W., Glushka, J., van    Halbeek, H., and Alhadeff, J. A. (1993) Glycobiology 3(3), 249-259-   38. Renkonen, O., Penttilā, L., Makkonen, A., Niemelā, R., Leppānen,    A., Helin, J. and Vainio, A. (1989) Glycoconjugate J., 6, 129-140.-   39. de Vries, T., Srnka, C. A., Palcic, M. M., Swiedler, S. J., van    den Ejnden, D. H. and Macher, B. A., (1995) J. Biol. Chem. 270,    8712-8722-   40. Niemelā, R., Natunen, J., Majuri, M. L., Maaheimo, H., Helin,    J., Lowe, J. B., Renkonen, O. and Renkonen, R. (1998) J. Biol.    Chem., 273, 4021-4026.-   41. Nagasawa, K., Inoue, Y. and Kamata, T. (1997) Carbohydrate Res.,    58, 47-55.

1. An oligosaccharide having the formula

wherein A is H or a glycosidically β1-3 linked D-glucopyranosyl residue(Glcβ1-3), R₁ is OH, R₂ is H and R₃ is OH, acylamido or —NH-acyl or R₁is H, R₂ is OH and R₃ is acetamido or —NHCOCH₃; B is H, an α-L-fucosylor an α-L-fucosyl analogue, and R₄ is OH, acetamido or —NHCOCH₃, thecurved line between the saccharide units indicating that themonosaccharide 1 is β1-4 linked to monosaccharide 2 when B is linked tothe position 3 of the monosaccharide 2, and the monosaccharide 1is β1-3linked to monosaccharide 2 when B is linked to the position 4 of themonosaccharide 2, monosaccharide 1 is GalNAc only when monosaccharide 2is Glc, n is 1 to 100, with the proviso that there is always at leastone α-fucosyl or α-fucosyl analogous group present in the molecule, andp and k are 0 or 1, and 1≦m≦1000, X is a monosaccharide selected fromthe group consisting of Glc, GlcNAc, Gal or GalNAc, optionally inreduced form, or oligosaccharide containing one or more of saidmonosaccharide units, the monosaccharide 2 being β1-3 or β1-4 linked tosaccharide X, Y is a spacer or linking group capable of linking X to Z,and Z is a mono- or polyvalent carrier molecule.
 2. The oligosaccharideaccording to claim 1, wherein B is α-L-fucosyl.
 3. The oligosaccharideaccording to claim 2, wherein monosaccharide 1 is Glc or GlcNAc.
 4. Theoligosaccharide according to claim 1, wherein m is 1 to 100, and n is 1to
 10. 5. The oligosaccharide according to claim 4, wherein m is 1 to10.
 6. The oligosaccharide according to claim 1, wherein they have theformula

wherein the symbols have the meanings given in connection with theformula I in claim 1, and wherein the monosaccharides 1 and 2 areindependently Glc and GlcNAc, B is L-fucosyl, and X is Glc or GlcNAc ora β1-3 or β1-4 linked oligomer comprising up to 10 units of Glc and/orGlcNAc.
 7. The oligosaccharide according to claim 1, wherein A is H andthe monosaccharides 1 and 2 are independently Glc or GlcNAc, B isL-fucosyl, p and k=0 and n=m=1, and X is Glc or GlcNAc or a β1-3 or β1-4linked oligomer comprising up to 10 units of Glc and/or GlcNAc havingthe formula


8. The oligosaccharide according to claim 1 having the formulaGlc/GlcNAcβ1-4(Fucβ1-3)Glc/GlcNAc(β1-4Glc/GlcNAc)_(n′) wherein n′ is theinteger 1 to
 8. 9. The oligosaccharide according to claim 8, wherein n′is the integer 1 to
 6. 10. The oligosaccharide according to claim 1having the formulaGlcNAcylβ1-4(Fucα1-3(GlcNAc(β1-4GlcNAc)_(n′) wherein n′ is the integer 1to 8 and acyl is an alkanoyl group which contains 8 to 24 carbon atomsand 1 to 3 double bonds.
 11. The oligosaccharide according to claim 1having the formulaGlcβ1-(-3GalNAc/GlcNAcβ1-4(Fucα1-3/H)Glcβ1-)_(n)-3GalNAcol/GlcNAcolwherein 1≦n≦1000.
 12. A process for the preparation of anoligosaccharide having the formula

wherein A is H or a glycosidically β1-3 linked D-glucopyranosyl residue(Glcβ1-3), R₁ is OH, R₂ is H and R₃ is OH, acylamido or —NH-acyl or R₁is H, R₂ is OH and R₃ is acetamido or —NHCOCH₃; B is H, an α-L-fucosylor an α-L-fucosyl analogue, and R₄ is OH, acetamido or —NHCOCH₃, thecurved line between the saccharide units indicating that themonosaccharide 1 is β1-4 linked to monosaccharide 2 when B is linked tothe position 3 of the monosaccharide 2, and the monosaccharide 1 is β1-3linked to monosaccharide 2 when B is linked to the position 4 of themonosaccharide 2, monosaccharide 1 is GalNAc only when monosaccharide 2is Glc, n is 1 to 100, with the proviso that there is always at leastone α-fucosyl or α-fucosyl analogous group present in the molecule, andi) p and k are 0 and m is 1, in which case X is H, an aglycon residue ora monosaccharide selected from the group consisting of Glc, GlcNAc, Galor GalNAc, optionally in reduced form, or oligosaccharide containing oneor more of said monosaccharide units, the monosaccharide 2 being β1-2,β1-3, β1-4 or β1-6 linked to saccharide X, with the proviso that X isnot H when both monosaccharides 1 and 2 are GlcNAc, B is L-fucosyl and nis 1 or ii) p is 1, k is 0 or 1 and 1≦m≦1000, in which case X is astraight bond, or a mono- or oligosaccharide as defined under i), Y is aspacer or linking group capable of linking the saccharide 2 or X is Z,and Z is a mono- or polyvalent carrier molecule, said process beingcharacterized in that a compound of the formula I, wherein B is alwaysH, is fucosylated with donor nucleotide sugar containing L-fucose, or ananalogue thereof, in the presence of a fucosyl transferase enzyme, andthe fucosylated saccharide so prepared is optionally recovered.
 13. Theprocess according to claim 12, wherein the fucosyltransferase ismammalian α1-3 or α1-3/4 fucosyltransferase.
 14. The process accordingto claim 12 or 13, wherein a N-acetyl-chitooligosaccharide is used asthe starting material.
 15. The process according to claims 12 or 13,wherein the donor nucleotide sugar containing L-fucose is GDP-L-fucose.16. The process according to claim 13, wherein the fucosyltransferase ishuman α1-3 fucosyltransferase or α1-3/4 fucosyltransferase III-VII, IXor α1-3/α1-3/4 fucosyltransferase of human milk.
 17. The processaccording to any one of claim 12, 13 or 16, wherein it comprises thefurther step of reacting the product obtained with the formula I withβ-N-acetyl-hexosaminidase.
 18. The oligosaccharide of claim 1, whereinmonosaccharide 1 is Glc.
 19. The oligosaccharide of claim 1, whereinmonosaccharide 1 is GlcNAcyl.
 20. The oligosaccharide of claim 1,wherein monosaccharide 1 is GalNAc.
 21. The oligosaccharide of claim 1,wherein monosaccharide 2 is optionally fucosylated Glc.
 22. Theoligosaccharide of claim 1, wherein monosaccharide 2 is optionallyfucosylated GlcNAc.
 23. The process according to claim 12, whereinmonosaccharide 1 is Glc.
 24. The process according to claim 12, whereinmonosaccharide 1 is GlcNAcyl.
 25. The process according to claim 12,wherein monosaccharide 1 is GalNAc.
 26. The process according to claim12, wherein monosaccharide 2 is optionally fucosylated Glc.
 27. Theprocess according to claim 12, wherein monosaccharide 2 is optionallyfucosylated GlcNAc.